Sickle cell disease (SCD) is a genetic disease that affects millions of people worldwide, with significant morbidity and a median life expectancy in the mid-forties. Although SCD can be cured by allogeneic hematopoietic stem cell transplantation (HSCT), this treatment strategy has substantial limitations and is only available to ~15% of patients. We have developed a genome-editing based strategy for treating SCD by correcting the sickle mutation in ?-globin (HBB) gene in patient?s hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 and corrective single-stranded oligonucleotide (ssODN) donor template, demonstrated that up to ~37% of mutant HBB alleles can be gene corrected. Injection of gene-edited SCD HSPCs into immunodeficient NOD/SCID/IL-2rgnull (NSG) mice showed a clinically relevant level of engraftment. We further demonstrated that cells differentiated from gene-edited SCD HSPCs produced high levels of normal hemoglobin A (HbA), resulting in a significant reduction of the amount of sickle hemoglobin (HbS) present in the red blood cells. In particular, delivery of Cas9/gRNA RNP into SCD CD34+ cells without ssODN template (i.e. only with Cas9 cutting of HBB) resulted in a large increase in fetal hemoglobin (HbF) induction and significant decrease in the amount of HbS, leading to prevention of sickling even under hypoxic conditions. However, the mechanism underlying HbF induction by Cas9 cutting is poorly understood, the clinical implications of large deletions/insertions at the HBB on-target cut-site and chromosomal rearrangements need to be determined, and the risk of inducing ?-thalassemia by HBB indels needs to be evaluated. The central hypothesis of the proposed research is that a quantitative understanding of HBB gene editing consequences will increase the efficacy and safety of gene-editing based treatment of SCD. In Aim 1 studies we will determine the mechanism(s) of Cas9-cutting induced HbF induction in SCD HSPCs by assessing the effect of Cas9 cutting of HBB on HSPCs in erythroid culture, and measuring the impact on relative expression of HBB and HBG. In Aim 2 we will quantify large deletions at HBB on-target site and chromosomal rearrangements in SCD HSPCs using new PCR and next-generation sequencing tools. In Aim 3 we will determine the potential of inducing ?-thalassemia due to HBB gene editing in SCD HSPCs by quantifying the total hemoglobin protein levels and the complete hemoglobin profile using our sickle HUDEP-2 cell-line and cells from gene-edited SCD HSPCs, and engrafted edited cells in a sickle mouse model. These studies will facilitate the translation of genome editing based SCD treatment into clinical practice.